The magnitude of CO2 countercurrent diffusional trapping (net diffusion from coronary venous to coronary arterial vessels) in the myocardium was determined. The left main coronary artery was pump perfused with a modified Gregg cannula in open-chest, heart-blocked, anesthetized dogs. Coronary venous PCO2, pH, and PO2 were simultaneously measured with a rapidly responding continuous electrode-cuvette system. Coronary venous CO2 concentration was calculated from the electrode outputs and a corresponding hemoglobin determination. After a step change in coronary flow, the amount of CO2 washed out or retained by the perfused myocardium was determined from the resulting coronary venous CO2 concentration changes. Analysis of the data with a compartmental mass balance model indicated that CO2 washouts and retentions were best fit with a model in which 80% of the myocardial CO2 resides in the interstitial and intracellular compartments and the remaining 20% in vascular compartments. The maximal diffusional trapping compatible with the data resulted in a modest end-capillary-to-end-venous CO2 concentration ratio of 1.02 to 1.04 at resting coronary flows. It is concluded that diffusional trapping of myocardial CO2 was below the detection limits of the experiment and that at normal coronary blood flow rates there is little CO2 diffusional trapping.